Wills J W, Craven R C, Achacoso J A
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center-Shreveport 71130-3932.
J Virol. 1989 Oct;63(10):4331-43. doi: 10.1128/JVI.63.10.4331-4343.1989.
Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.
劳氏肉瘤病毒（RSV）是逆转录病毒禽肉瘤和白血病家族的成员，长期以来人们一直知道它能够感染并转化哺乳动物细胞；然而，这种转化后的细胞不会释放病毒颗粒。在这些细胞中产生的RSV gag产物（Pr76gag）不会释放到培养基中，也不会经过蛋白水解加工以释放成熟产物。因此，Pr76gag在哺乳动物细胞中的行为与哺乳动物逆转录病毒Gag蛋白非常相似，后者已被改变以阻止在第2位残基（甘氨酸）处添加肉豆蔻酸。由于RSV gag产物不具备肉豆蔻酸添加位点，我们推测通过寡核苷酸定向诱变创建一个这样的位点可能会使颗粒从哺乳动物细胞中释放出来。我们创建了两种肉豆蔻酰化形式的Pr76。在Pr76myr1中，前10个氨基酸已被替换为p60v-src的氨基酸，已知这些氨基酸足以进行肉豆蔻酰化。在Pr76myr2中，第二个残基处的谷氨酸已被甘氨酸取代。通过使用基于SV40的载体，编码修饰型和野生型Pr76的等位基因已在哺乳动物（CV-1）细胞中高水平表达。令人惊讶的是，我们发现高水平表达未修饰的（野生型）产物Pr76myr0会导致颗粒形成和前体加工水平较低。这表明肉豆蔻酸不是靶向作用的唯一决定因素。然而，向Pr76myr1或Pr76myr2添加肉豆蔻酸会使Gag功能增强五倍。在所研究的所有方面，肉豆蔻酰化的Pr76的行为与在禽类细胞中产生的真实产物相同。我们还表明，加工是由gag编码的蛋白酶介导的，去除氨基末端以产生Pr76gagX会导致无法形成颗粒或进行加工。这表明正确的靶向作用是RSV蛋白酶在哺乳动物细胞中激活的先决条件。
